Multidrug resistance (MDR) remains one of the major obstacles to successful chemotherapy. Although the study of MDR in the laboratory and in the clinic has progressed at an impressive rate within the past several years, several critical questions remain unanswered. Little is known about the mechanism(s) regulating the genes that encode P-glycoprotein, the putative drug efflux pump whose overexpression is, at least in part, responsible for the resistant phenotype. Our laboratory is attempting to define these mechanisms in normal cells during development and differentiation as well as during the development and maintenance of the multidrug-resistant phenotype. We propose to examine these mechanisms, beginning with a careful dissection of the components required for the constitutive expression of the hamster pgp1 gene. Our recent identification of a differential utilization of transcription start sites between drug-sensitive and drug-resistant cells suggests a mechanism for the overexpression of pgp1 in the absence of gene amplification in actinomycin D (ActD)-selected cells; a long-range goal of our studies is to define such a mechanism and determine the transcriptional or post- transcriptional factors underlying the increased expression of the pgp1 gene in MDR cells. The experiments outlined in Specific Aim 1 are directed at defining the DNA sequence elements and protein factors required for the constitutive expression of the hamster pgp1 gene. Ultimately, we hope to identify the DNA and protein factors involved in the tissue-specific expression of pgp1, using hamster liver and hepatocytes as a model system. Furthermore, we will investigate the mechanism underlying the differential utilization of pgp1 transcription initiation sites. In Specific Aim 2, we will further dissect the transcriptional regulation of the pgp1 gene by examining its expression in a cell-free transcription system, with a long-range goal of reconstituting tissue-specific transcription in vitro. In Specific Aim 3, we propose to investigate the role that post-transcriptional mechanisms play in the regulation of pgp1 expression in drug-sensitive vs. drug- resistant cells.